AU598463B2 - Duboisia hybrid cultivar - Google Patents

Description

.fk 598463 COMMONWEALTH OF AUSTRALIA Patents Act 1952 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number Lodged Complete Specification Lodged Accepted Published Priority Related Art This document contains the amendments made under Section 49 and is correct for printing.

494* 4 4 9 4 4 i 4 Name of Applicant Address of Applicant Actual Inventor Address for Service BOEHRINGER INGELHEIM INTERNATIONAL GmbH Postfach 200 D-6507 Ingelheim am Rhein German Federal Republic S Dr. Hartmut Groeschel Veit-Stoss-Str. 17 D-6507 Ingelheim F.B. RICE CO., Patent Attorneys 28A Montague Street BALMAIN NSW 2041 4 4 Complete Specification for the invention entitled: DUBOISIA HYBRID CULTIVAR The following statement is a full description of this invention including the best method of performing it known to us:- *4 4* 4 ii j~

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INTRODUCTION

This invention relates to a new Duboisia hybrid cultivar of a mixed hyoscine/hyoscyamine type, denominated "H 38/3".

The native Australian plant species Duboisia myoporoides R.Br., Duboisia leichhardtii F.v. Muell., and interspecific hybrids of these, all known under the common name "corkwood", are commercially exploited for the production of alkaloid drug.

The interspecific hybrids of Duboisia myoporoides and Duboisia leichhardtii predominantly cultivated commercially are descendants from hybrids obtained from crosses executed by Australian government agencies before 1950, or were obtained through selection from spontaneous interspecific crosses produced accidentally within large Duboisia ssp.

populations.

Thel probability that plants with meritorious value are found is low since the combination of parents hybridized, as I" well as the selection take place accidentally.

V 20 Contrary to that, the new hybrid subject of this invention was obtained by controlled hybridization of selected parents, combined with systematic screenings at A* different stages for vigour of growth and chemical composition, resulting in a plant with a combination of desired characteristics.

To establish a cultivar with meritorious value, t*i, thousands of plants have to be raised in nurseries and kept S, for years, screened for alkaloid content by costly analytical methods, artificially cross-pollinated in 30 painstaking handwork, and finally field tested for several years.

*Accordingly it is a substantially valuable discovery taking much skill in breeding techniques and careful screening and testing as well as substantial luck to result in a meritorious Duboisia hybrid cultivar.

rIV r. 3 CHARACTERIZATION OF DUBOISIA HYBRIDS Botanical Characters 0944 04r .4 9 4,, *0P *0 0d The species Duboisia myoporoides and Duboisia leichhardtii are well distinguishable morphologically, while their hybrids may show slight differences only with respect to shape of flowers and leaves, making distinction difficult, if not impossible, even for the trained.

Interspecific hybrids D. myoporoides x D. leichhardtii have the following characteristics: Tall shrub or small tree, glabrous, bark thick and corky towards base. Leaves alternate, simple, narrowly ovate to narrowly obovate, almost sessile or with petiole to 3 cm long, acuminate to obtuse, 4-15 cm long 0,7-4 cm wide, slightly discolorous to concolorous. Inflorescence paniclelike, broadly pyramidal, terminal, leafy.

Flowers bisexual, slightly zygomorphic, subtended by pairs of opposite bracts 0.5-11 mm long; pedicels 2-16 mm 20 long. Calyx regular, campanulate, 5-lobed, 1-3 mm long, the tobes on-fifth to half as long as tube.

Corolla campanulate, white with purple striations in throat, 6-15 mm long; tube funnel-shaped to campanulate, 3mm diam. at apex; limb 5-lobed, lobes 3-8 mm long, round to acute, volutive in bud. Stamens 4, sometimes didynamous, inserted at base of corolla tube, 1.5-4 mm long; a staminode sometimes present; anthers unilocular, not cohering, dehiscing by a terminal, semicircular slit. Ovary bilocular; style 1.5-4 mm long, about as long as upper stamens; stigma capitate, very shortly bilobed. Fruit a succulent berry, globose, rarely ellipsoid to ovoid, 4-8 mm long, purple-black, fruiting pedicels 8-18 mm long. Seeds reniform, 2.5-3 mm long. 4

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4..t* 0) 4, 0 4 'In 4 Morphologically, Duboisia hybrid flowers are intermediate between those of D. myoporoides and D.

leichardtii, the differences being gradual ones only so that they are difficult to describe unmistakably for identification purposes.

Leaf shape, however, can be measured physically, and there are stable, statistically significant differences between natural species and hybrid clones. The leaf shape of Duboisia species and their hybrids can be described by their leaf shape index, which is determined by bisecting the leaf transversely and expressing the area of the distal section as a fraction of the area of the proximal section.

The leaf shape index for Duboisia myoporoides is about 1.7, that for Duboisia leichhardtii about 1.3.

(Reference: Groszmann, Keleny, and Rodwell, C.N.

1949.Hybrids between Duboisia myoporoides and D.

leichhardtii. Qld.J. Agric. Sci. Vol.6, No.3.) Chemotypical Characters E *e 1, 20 t, Total amount as well as proportion of alkaloids V contained in Duboisia leaves vary with the age of the plant, season of the year, actual weather conditions, soil water conditions, and nutrient supply, just mentioning the main factors. Therefore, only the prevailing range of variation of the main alkaloids in the dry leaf of mature trees (age 12 months at least) during the usual harvesting period (October to March, approximately) may serve for approximative characterization.

S 30 The range of content of the "standard" hybrid (definition see below) is 1.0 to 2.3 hyoscine (base) and 0.0 to 0.6 hyoscyamine (base). Duboisia myoporoides leaf icommercially collected from the wilds in 1966/67 had a content of 0.36 hyoscine (base).

5 Duboisia leichhardtii leaf collected from the wilds contained 0.5 hyoscine (base) and up to 1,5 hyoscyamine (base) approximately.

While alkaloid content due to its variability is of limited use only for characterization, it has been found that a specific Duboisia cultivar can be uniquely identified by its leaf tissue peroxidase pattern assessed by isoelectric focusing.

Assessment of the proteine or enzyme pattern of tissue from determined plant organs by electrophoresis or isoelectric focusing is widely used for cultivar identification, especially of those cultivars under plant variety rights, where phenotypical characteristics are not sufficient for unequivocable distinction.

For example, these techniques are being used in Australia by CSIRO for plant breeding purposes where no suitable morphological markers exist.

(Reference: HAYWARD, M.D. and J.B. Hacker, 1980. Genetic Scontrol of some esterase isozymes of DIGITARIA species, and S' 20 their utility in the identification of hybrids. Euphytica 29: 347-356).

r' The procedure for assessment of the leaf tissue S< peroxidase pattern is shortly described below: From mature, fresh or deep-frozen leaves of adult p,.ants cell sap is extracted, and by common biochemical techniques the peroxidase enzymes are isolated.

The solution containing the enzymes is then applied to a 0 a thin or ultra-thin polyacrylamide gel layer.

Through electrodes on the sides of the gel sheet the enzymes 30 are exposed to an electric tension, which after a determined time results in a dislocation typical for each of the enzymes present.

Through staining and subsequent densitometric scanning of the resulting banding pattern a graph is obtained which indicates type and relative quantity of peroxidase enzymes present in the leaf sample tested.

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6- DUBOISIA YIELDS From the age of at least 12 months on, Duboisia plants are usually harvested once a year. Their productive life span is 5 to 12 years, depending on growth conditions and agricultural practices. The parts harvested are all branches cut at about 50 cm above ground. The commercial drug consists in the dried leaf removed from the branches.

The dry leaf yields vary in a wide range from about 0.6 to 1.8 t/ha, dependent on soil, weather, and agricultural practices. The normal range of yields of the "standard" hybrid as well as of the new cultivars is from 0.8 to 1.2 t/ha. (Exact data on Duboisia hybrid yields are not cited in literature, nor are they disclosed by the growers.) Alkaloid content and dry leaf yield are correlated nearly reciprocally. Accordingly, "standard" hybrid yields in terms of pure alkaloid amount to 15 20 kg/ha hyoscine (base) and kg/ha hyoscyamine (base).

For Duboisia myoporoides and Duboisia leichhardtii yields no such data are available, since leaf of the native Duboisia species has always been collected by individual collectors from trees dispersed in the native forests of its C habitat only, and no yield records exist. To our knowledge, Duboisia myoporoides was never grown in commercial plantations.

SCOMMON DUBOISIA HYBRID CULTIVARS The Duboisia hybrids predominantly cultivated in the 0' 30 Duboisia growing area in Queensland are descendants from the early crosses obtained by Australian government agencies before 1950. They are commonly called "standard hybrids" or Sp "standards". Rooted cuttings of "standard" hybrids are available to the public from commercial nurseries.

4- 7 Other Duboisia hybrid cultivars are known in some cases to be grown by their breeders, individual growers who do not disclose specific data. However, the alkaloid content of the drug commercialized rarely exceeds that of the "standard" hybrid.

PROPAGATION OF DUBOISIA HYBRIDS Duboisia hybrids are usually mass propagated by individual growers for own use, or by commercial nurseries.

Propagation is done by cloning using cutting techniques commonly known to nursery professionals and specialized growers.

EXTRACTION OF ALKALOIDS The extraction process is commonly known in the pharmaceutical industry. It is described for example in F. E. HAMERSLAG: The technology and chemistry of alkaloids; D. van Nostrand Comp. Inc., Toronto, New York, London, 1950.

C C Cr t(4 8 OBTAINMENT OF THE NEW DUBOISIA CULTIVAR The best method known to the applicant of performing the invention, i. e. the plant breeding procedure which resulted in the new Duboisia hybrid cultivar subject of this application, is completely described below.

A Duboisia hybrid cultivar meritorious with respect to its hyoscine yield has been discovered resulting from hybridization of the parents M 3/1, a Duboisia myoporoides individual selected (and cloned subsequently) from the genuine Duboisia myoporoides population in New South Wales and Queensland (specifically from the Proston area of South Burnett, Queensland), and H 1/25, an interspecific D. myoporoides D. leichhardtii hybrid selected from the common Duboisia hybrid population in the Duboisia growing area of South Burnett, Queensland. The new hybrid was obtained in the following manner: a) From the natural Duboisia myoporoides population an individual was selected. Selection criteria were hyoscine content and vigour of growth.

b) From the common (commercial) "standard" hybrid population in the Duboisia growing Proston area of South Burnett, Queensland, individuals were selected.

Selection criteria were hyoscine and hyoscyamine content, vigour of growth and foliage density.

30 c) The selected parent plants were crossed artificially, the "standard" hybrid plants being the pollen fathers.

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-9d) The F 1 generation resulting from the seeds obtained from the crossings (300 plants approximately) was screened for vigour of growth, tree shape (broad, ramified, strong trees preferred against tall, thin, monoaxial ones), density of foliage, leaf size, hyoscine and hyoscyamine content.

e) About 40 individual F 1 plants selected were cloned by vegetative propagation using common cutting techniques familiar to nursery pr.fe.sionals. Here, selection forooting behaviour took place.

f) The remaining hybrid clones underwent a first field test with subsequent selection according to the criteria listed under d).

g) The 10 best hybrid clones then were propagated m oo*# vegetatively and planted in the piduction area for a o0: second field test. In the course of 4 years, 20 harvesting once a year, the clones were further :00• selected applying the same criteria as before, plus yield, insect pest resistance, storm resistance, and 0 e• U .regrowing behaviour.

h) For final relection, the best clones from g) were bulked up for establishment of a pilot plantation of several hectares. After 2 3 harvests, the new hybrid clone H 38/3 was chosen for commercial production.

REPEAt4BILITY OF THE INVENTION As most chemical and biological processes, the complex breeding procedure described before is not 100 repeatable.

However, the Duboisia ssp. populations from which tbd p1

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r mi,, 1 i i L~i--url Cw--i _rr-~ 10 breeding procedure started are clearly identified and readily accessible to the interested.

The techniques involved are commonly known. Thus, any person skilled in the art can repeat the procedure following the description of the best method of performing the invention.

Provided that a similar effort is made over a similar period of time, there is a probability of more than 95 that an equally meritorious hybrid clone with similar characteristics will be obtained.

Luck is involved insofar as the time needed to achieve the desired goal may be shorter or longE.r.

DESCRIPTION OF THE NEW DUROISIA CULTIVAR The invention the subject of this specification can then be said to reside in the Duboisia hybrid cultivar denominated H 38/3 as identified hereinafter by being the following: 20 Morphology Morphological characteristics correspond to the general botanical description of Duboisia hybrids given above.

The flowers of the new hybrid are morpholoqg ally i-termediate between the flower of D. myoporoides and D. leichhardtii, i. e. the corolla lobes are longer, wider and more acuminate than those of D. myoporoides, but shorter, less narrow and less acuminate than those of D. leichhardtii.

The accompanying colour photographs show foliage and inflorescences of the new cultivar.

Leaf shape index for the new hybrid subject of this invention is 1.44, while that of its parents is 1.7 (Duboisia myoporoides clone M 3/1) and 1.50 (Duboisia hybrid clone H 1/25), respectively.

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I 11 Genotype Progeny F 1 from hybridization of the Duboisia myoporoides clone M 3/1 as mother, and the Duboisia hybrid clone H 1/25 as father.

Alkaloid content The average hyoscine content of leaves of the cultivar H 38/3 equals or exceeds the average content of the high yielding varieties cultivated at present, mainly the "Standard" hybrid descendant from the early hybridization experiments mentioned above, while the hyoscyamine content which is negligible in the "Standard" hybrid is about the same as the hyoscine content at the time when harvested nsually.

Prevailing range of variation of the main alkaloids in the dry leaf of mature trees (age 12 month at least) during the usual harvesting period (October to March, approximately): Duboisia hybrid cultivar H 38/3 hyoscine (base) 1,4 2,3 hyoscyamine (base) 1,0 2,2 as opposed to the parents: Dtboisia myoporoides clone M 3/1 hyoscine (base) 0,94 hyoscyamine (base) 0,37 (means of 10 years of experimental Si cultivation) SDuboisia hybrid clone H 1/25 hyoscine (base) 2,6 hyoscyamine (base) 0,7 ii;ri-i -r i 12 and to the populations where the parents were selected from: Duboisia myoporoides leaf commercially collected from the wilds (1966/67) hyoscine (base) 0,36 "standard" hybrid hyoscine (base) 1,0 2,3 hyoscyamine (base) 0,0 0,6 Alkaloid yield Duboisia hybrid H 38/3: hyoscine (base) 16 20 kg/ha hyoscyamine (base) 14 20 kg/ha as opposed to "standard" hybrid hyoscine (base) 15 20 kg/ha hyoscyamine (base) 0 5 kg/ha (Parent clones M 3/1 and H 1/25 were propagated for breeding purposes in small numbers only; no yield assessed.) Leaf tissue peroxidase pattern The specific peroxidase patterns of the Duboisia hybrid cultivar H 38/3 subject of this specification, and of the parent clones Duboisia myoporoides M 3/1 and Duboisia hybrid H 1/25 are shown in a photograph of the respective i 30 polyacrylamide layer (Fig.l).

The graphs resulting from densitometric scanning of the banding patterns in Fig. 1 are shown in Fig. 2, 3 S" S and 4.

f n 4U 13 Fig.l. Ultra-thin polyacrylamide layer isoelectric focusing of leaf tissue peroxidases from Duboisia hybrid H 38/3 and its parents.

Fig.2. Densitometric scanning of the peroxidase banding pattern of Duboisia hybrid cultivar H 38/3.

Fig.3. Densitometric scanning of the peroxidase banding pattern of Duboisia myoporoides clone M 3/1.

Fig.4. Densitometric scanning of the peroxidase banding pattern of Duboisia hybrid clone H 1/25.

The procedure followed when preparing the peroxidase.

patterns is as follows: Materials and method sample preparation: Fresh or deep-frozen leaves, with a sand abrasive and a buffer, are ground to a fine paste.

S. Buffer solution: 0,1 mol tris(hydroxymethyl)aminomethane hydrochloride pH 7,2 100 mg sodium sulfite mg sodium thiosulfate 1 g Triton X 100

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2 0 ad 100 ml Per 1 g of leaf 1,8 ml buffer solution are added.

The paste is centrifuged for 1 h at 2 000 x g, and the supernatant filtered through a sterile filter with 0,22 Am membrane.

S 30 Isoelectric focusing: Horizontal thin layer isoelectric focusing by the method of GOERG et al. is employed.

The polyacrylamide gel (T 6,8 C 2,6 gel -14 thickness 0,48 mm) contains 3,8 Servalyt TR (1 part pH 2- 4, 3 parts pH 2'11) and 6 mol/l urea.

As carrier for the polyacrylamide gel, polyester foil (Gel-fixR Polyesterfolien, Serva, Heidelberg, FRG) is used.

Distance of separation: 9.6 cm.

Anode: Filter paper strip soaked with phosphoric acid Kathode filter paper strip soaked with ethylendiamine 2 Samples are pipetted into slots (10 x 2 x 0,3 mm) in the gel layer. The distance to the kathode is cm.

Separation is finished at a voltage of 1500 V and a current of 7 mA.

Peroxidase staining: Staining solution: 200 ml phosphate buffer 0,1 mol pH 7,2 g o-tolidine 0,3 ml hydrogen peroxide 30 200 ml of the above solution is poured over the gel slab placed in a trough. Bands develop after 30 seconds already.

The reaction is stopped by a methanol/H 2 0/glacial acetic acid mixture 45 45 10, the gel then air-dried.

Literatureg GOERG, POSTEL, WESTERMEIER, R. 1979: S 30 Ultradinnschicht-iscelektrische Fokussierung und Ultradfnnschicht-Elektrophorese in Polyacrylamidgel au£ Folie.

St* GIT Labor-Medizin 2: 32-40.

i AL: a^ 4i 4,-

Claims (4)

1. A new and distinct Duboisia hybrid cultivar, "H 38/3", as hereinbefore described and illustrated, identified especially by the following: Chemotype characterized by A. Leaf tissue peroxidase pattern assessed by ultra-thin polyacrylamide layer isoelf-tric focussing, and documented in Fig. 1 and Fig. 2. B. Amount and proportion of tropane alkaloids contained in the leaves, the hyoscine content being similar to or exceeding that of the "standard" hybrid, and the hyoscyamine content about the same as the hyoscine content at usual harvest time. Progeny F 1 from hybridization of the Duboisia myoporoides clone M 3/1 as mother, and the Duboisia hybrid clone H 1/25 as father. foa* f Leaf shape index (according to GROSZMANN, H.M. et al. 1949) 1.44.

2. Reproduction of the Duboisia hybrid cultivar to** identified in claim 1 by any means of vegetative or generative propagation. S<o

3. Utilization of leaf of the Duboisia cultivar identified in claim 1 for extraction of hyoscine and/or hyoscyamine.

4. Hyoscine and/or hyoscyamine when extracted from a eIe Duboisia cultivar hybrid as claimed in claim 1. the Applicant: a DATED this 29th day of March 1990 .aH^ *BOEHRINGER INGELHEIM Sa" INTERNATIONAL GmbH Patent Attorneys for the Applicant: F.B. RICE CO. C </9 'N i